New Members of the Centrapalus Coumarin and Pauciflorin Series from Centrapalus pauciflorus

Monoterpene and 5-methylcoumarin- or 5-methylchromone-coupled meroterpenoids occurring mainly in the Asteraceae species proved to have high potency against protozoans, worms, and various tumor cells, which make them interesting targets for searching for new bioactive compounds. The African plant Centrapalus pauciflorus was applied in traditional medicine for healing chest pain and stomach aches. Three new meroterpenoids named centrapalus coumarin N (2), pauciflorins P (3), and Q (4), and the already known cyclohoehnelia coumarin (1), were isolated from the chloroform extract of C. pauciflorus, together with centrapalus coumarin O (5), which was obtained for the first time from a natural source. The structures were established from HRESIMS, 1D (1H NMR, 13C NMR JMOD) and 2D NMR (HSQC, HMBC, 1H-1H COSY, NOESY) spectroscopies, and the absolute stereochemistry of 5 was determined by single-crystal X-ray diffraction. Compounds 1, 2, and 5 are hybrid molecules of 5-methylcoumarin–monoterpene origin. Centrapalus coumarin N is the first example of meroterpenoids, where a monoterpene is fused with a coumarin and an acetophenone unit. Pauciflorins P and Q are dimeric meroterpenoid isomers. Centrapalus coumarins N and O were tested for antiproliferative activity against human adherent breast (MCF-7, MDA-MB-231), cervical (HeLa, SiHa), and ovarian (A2780) cancer cell lines, and were additionally included to obtain data concerning cancer selectivity. Both compounds exhibited moderate (IC50 > 10 µM) but selective activity against A2780 cells.


Introduction
The incidence of cancer is rising worldwide, making the development of more effective cancer treatments imperative from a medical standpoint.The lack of selectivity in currently available chemotherapeutic drugs is their biggest drawback, since it leads to severe side effects that frequently restrict their range of use.Therefore, the main focus of cancer research for many years has been the development of chemotherapeutical drugs with distinct modes of action that guarantee great selectivity [1].
The word meroterpenoid was introduced by Sir John W. Cornforth in an article on the biosynthesis of terpenoids [2].Meroterpenoids can be defined as hybrid natural products derived from mixed terpenoid and polyketide or non-polyketide biosynthesis.Their carbon skeletons are formed by intramolecular cyclizations and rearrangements, resulting in different macrocyclic or polycyclic structures with a variety of functional groups.Since the introduction of the term in 1968, there has been an explosion in the number of identified compounds [3].Meroterpenoids are widely produced by a diverse array of microorganisms, fungi, plants, and animals [4][5][6][7][8][9].Adducts of 5-methylcoumarins or 5-methylchromones with monoterpenes have a restricted occurrence.They are mainly found in members of the tribes Vernonieae, Nassauvieae, Onoserideae, and Mutisieae in the Asteraceae family, and occasionally from a few other taxa [10,11].Due to their restricted occurrence, only some information was published about their pharmacological activities.Among the coumarin-based meroterpenoids, 2 ′ -epicycloisobrachycoumarinone epoxide and cycloisobrachycoumarinone epoxide, isolated from Vernonia brachycalyx, exhibited activity against Leishmania major promastigotes and Plasmodium falciparum schizonts in vitro and demonstrated weak inhibition on the proliferation of human lymphocytes [12].Ethuliacoumarin A and isoethuliacoumarin A obtained from Ethulia conyzoides showed molluscicidal activity against Biomphalaria glabrata and Bulinus truncatus.In addition, ethuliacoumarin A was found to have cercaricidal and ovicidal potencies [13].Among the chromane/chromene meroterpenoids, rubiginosins A-G, reported from Rhododendron rubiginosum, have been documented to have cytotoxic effects on various tumor cell lines (A549, HCT116, SK-HEP-1, and HL-60) [3].Enantiomeric pairs of meroterpenoids with protein tyrosine phosphatase 1B inhibitory activity were reported in Rhododendron fastigiatum, with IC 50 values ranging from 40.9 to 47.0 µM [4].
The genus Centrapalus, belonging to the Asteraceae family, consists of nine species, which are mainly annual or perennial herbs.Synonym names of Centrapalus pauciflorus (Willd.)H.Rob. include C. galamensis Cass., Conyza pauciflora Willd., Vernonia afromontana R.E.Fr., and Vernonia pauciflora (Willd.)Less., etc. C. pauciflorus can be found predominantly in tropical African countries [14,15].It is a mainly unbranched, annual plant that grows 3-5 m height.In traditional African medicine, the leaves of C. pauciflorus are used as tea or cooked in porridge to ease chest pain and stomach aches [16].C. pauciflorus was noted as a potential source of vernolic acid, a potentially useful biofeed stock.Aliphatic and phenolic acids, flavonoids, coumarins, and sesqui-and triterpenoids were previously described to be present in C. pauciflorus [17].In our previous studies, 36 monoterpene-coupled meroterpenoids were isolated from the methanol extract of the leaves of C. pauciflorus, 28 of them were new natural compounds.The compounds belong partly to the centrapalus coumarin series that includes 5-metlylcoumarin adducts, and partly to the pauciflorin series that includes 5-methylchromone or 2,4-chromadione adducts.Among the compounds, centrapalus coumarins A, B, and F, and pauciflorin F showed remarkable antiproliferative activity (IC 50 < 10 µM) against human adherent breast (MCF-7) and cervical (HeLa) cancer cell lines.
In continuation of our ongoing study on C. pauciflorus metabolites [16,18,19], the present paper reports the isolation and structure determination of five meroterpenoids (1-5) from the chloroform phase of the MeOH extract of leaves (Figure 1).Compounds 1-4 are new natural products, while compound 5 was previously reported as a semisynthetic compound.Two compounds (2 and 5) were investigated for antiproliferative activity against human tumor cell lines of gynecological origin and a non-malignant cell line.

Plant Material
The plant collection and voucher specimen depositions are reported in ref. [17].

General Experimental Procedures
Optical rotations were determined with a Jasco P-2000 polarimeter (Jasco International Co., Ltd., Hachioji, Tokyo, Japan).NMR spectra were recorded in CDCl3 on a Bruker Avance DRX 500 spectrometer (Billerica, MA, USA) at 500 MHz

Plant Material
The plant collection and voucher specimen depositions are reported in ref. [17].

X-ray Crystallography of Centrapalus Coumarin O (5)
The absolute configuration of 5 was determined by X-ray crystallography.The single crystal was harvested from a solution prepared with the mixture of MeOH and ethyl acetate in a 3:2 ratio by evaporation.Single-crystal X-ray diffraction data collection was performed on well-developed, colorless, clear, transparent, block-type crystals of 5 using CuKα radiation.Compound 5 crystallizes in the orthorhombic crystal system, in the chiral space group P2 1 2 1 2 1 (#19).The absolute configurations of the chiral atoms are C-3 ′ (R) and C-5 ′ (S).Details of molecular conformation, intra-and intermolecular interactions, and packing arrangement are published as Supporting Information.
Crystallographic data of compound 5 have been deposited in the Cambridge Crystallographic Data Centre with the deposition number 2314286.Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, by emailing data_request@ ccdc.cam.ac.uk, or by contacting the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44-(0)1223-336033].

Determination of Antiproliferative Activities
The antiproliferative activities of the isolated compounds were investigated on a panel of human adherent cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay [22].Cell lines isolated from cervical (HeLa and SiHa), breast (MCF-7 and MDA-MB-231), ovarian cancers (A2780), and non-malignant mouse fibroblasts (NIH/3T3) were purchased from the European Collection of Cell Cultures (Salisbury, UK).The SiHa cell line was obtained from the American Tissue Culture Collection (Manassas, VA, USA).The cell culturing and determination of the antiproliferative effects of the isolated compounds were conducted using a methodology described previously [15].Calculations were performed using GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA, USA).
Centrapalus coumarin O (5), a white crystal, is a new natural product.Previously, it was semisynthetized from a compound of Ethulia conyzoides and obtained by methylation as a mixture of 5 ′ α and 5 ′ β isomers [24]. 1 H and 13 C NMR assignments of compound 5 were determined from 2D NMR spectra.The NMR data of 5 are listed in Table 3, because in a previous publication, only 1 H NMR chemical shifts were published for the mixture of the isomers.The absolute configuration of 5 was analyzed by single-crystal X-ray diffraction, and absolute configurations of C-3 ′ and C-5 ′ were established as R and S, respectively (Figure 4).The Flack parameter was 0.09 (6).The absolute configuration of 5 was analyzed by single-crystal X-ray diffraction, and absolute configurations of C-3′ and C-5′ were established as R and S, respectively (Figure 4).The Flack parameter was 0.09 (6).

Antiproliferative Activity of Centrapalus Coumarins N and O
The antiproliferative properties of centrapalus coumarins N (2) and O (5) were investigated against a panel of human adherent tumor cell lines of gynecological origin by in vitro MTT assay (Table 4).The amounts of the other compounds were insufficient for the in vitro assay.Compounds 2 and 5 were tested on the breast (MCF-7, MDA-MB-231), cervical (HeLa, SiHa), and ovarian (A2780) cancer cells in two concentrations (10 and 30 µM).The anticancer agent cisplatin was used as a positive control.The A2780 ovarian cell line appeared to be the most sensitive (growth inhibition > 50%); therefore, IC50 values were determined for the compounds (IC50 2: 20.11 µM, 5: 15.99 µM).Non-cancerous mouse fibroblasts (NIH/3T3) were additionally included to characterize the cancer selectivity of the tested compounds.Since compounds 2 and 5 elicited weak action against fibroblasts, lower than against the ovarian cancer cells, their effect detected on A2780 cells could be regarded as selective.

Antiproliferative Activity of Centrapalus Coumarins N and O
The antiproliferative properties of centrapalus coumarins N (2) and O (5) were investigated against a panel of human adherent tumor cell lines of gynecological origin by in vitro MTT assay (Table 4).The amounts of the other compounds were insufficient for the in vitro assay.Compounds 2 and 5 were tested on the breast (MCF-7, MDA-MB-231), cervical (HeLa, SiHa), and ovarian (A2780) cancer cells in two concentrations (10 and 30 µM).The anticancer agent cisplatin was used as a positive control.The A2780 ovarian cell line appeared to be the most sensitive (growth inhibition > 50%); therefore, IC 50 values were determined for the compounds (IC 50 2: 20.11 µM, 5: 15.99 µM).Non-cancerous mouse fibroblasts (NIH/3T3) were additionally included to characterize the cancer selectivity of the tested compounds.Since compounds 2 and 5 elicited weak action against fibroblasts, lower than against the ovarian cancer cells, their effect detected on A2780 cells could be regarded as selective.Since meroterpenoids are a structurally outstandingly diverse group of natural products, elucidating their structure-activity relationships is a great challenge.Based on our previous works, we can make a few statements regarding structure-activity relationships.Comparing the structures of 5-methylcoumarin derivative 6 (Figure 5) previously isolated from C. pauciflorus [16] and centrapalus coumarin O (5), it can be observed that, in position 5 ′ , compound 5 contains a C 4 unit 2-carboxymethyl-2-methyl-1-propenyl group, which forms a lactone ring in 6.Since the antiproliferative activity of 6 is more pronounced on MCF-7 (IC 50 15.30µM), HeLa (IC 50 14.59 µM), and SiHa (IC 50 18.94 µM) cells than those of 5, it can be stated that the lactone structure at C-5 ′ is preferable for the effect on breast and cervical cancer cells.However, compound 5 exerted greater activity against ovarian cancer cells (A2780) (IC 50 15.99 µM) than 6 (IC 50 of 29.94 µM).Centrapalus coumarin A (7) with the α-dimethylvinyl group at C-5 ′ showed a similar effect against the ovarian cancer cell line (IC 50 19.65 µM) as 5; in addition, it was also potent against HeLa cells (IC 50 of 9.21 µM).
Centrapalus coumarin N (2) contains a 5-methylchromone and a 5-methylcoumarin part; the latter structure can also be found in the C. pauciflorus metabolite Hoehnelia coumarin (8), where it is connected to a methyl-dihydrofuranolactone ring.As the Hoehnelia coumarin (8) was ineffective in our previous antiproliferative assay [16], the activity of centrapalus coumarin N (2) against A2780 ovarian cancer cells (IC50 20.11 µM) most probably can be attributed to the 5-methylchromone part of the molecule.Furthermore, it was observed that a hydroxy function on the aromatic ring may substantially increase the antiproliferative activity.This was found when the antiproliferative activities of pauciflorin F and pauciflorin G were compared [19].A set of rubiginosins and anthopogochromene B, all molecules containing a hydroxy group on the coumarin core, Centrapalus coumarin N (2) contains a 5-methylchromone and a 5-methylcoumarin part; the latter structure can also be found in the C. pauciflorus metabolite Hoehnelia coumarin (8), where it is connected to a methyl-dihydrofuranolactone ring.As the Hoehnelia coumarin (8) was ineffective in our previous antiproliferative assay [16], the activity of centrapalus coumarin N (2) against A2780 ovarian cancer cells (IC 50 20.11µM) most probably can be attributed to the 5-methylchromone part of the molecule.Furthermore, it was observed that a hydroxy function on the aromatic ring may substantially increase the antiproliferative activity.This was found when the antiproliferative activities of pauciflorin F and pauciflorin G were compared [19].A set of rubiginosins and anthopogochromene B, all molecules containing a hydroxy group on the coumarin core, were reported to exert moderate cytotoxic activity against four cancer cell lines [25].In addition to the above, the compounds are worth investigating further.Their other activities reported for monoterpene-based meroterpenoids include antioxidant, antimicrobial, and various enzyme inhibitory effects [4].

Conclusions
The chloroform phase of the methanol extract from C. pauciflorus leaves underwent a multistep isolation process with the goal of identifying new plant metabolites with potential antiproliferative activity.Five compounds (1-5) were reported in the present paper that represent structural novelties: centrapalus coumarins N (2) and O (5) isolated from C. pauciflorus are hybrid molecules with 5-methylcoumarin-monoterpene origin.Centrapalus coumarin N (2) is the first example of a meroterpenoid where the monoterpene is fused with a coumarin and an acetophenone unit.Pauciflorins P (3) and Q (4) are dimeric meroterpenoid isomers with a symmetric structure.
The identified structures allow the recognition of some biogenetic relationships.Compounds 3 and 4 can be derived by coupling two pauciflorin N molecules, after rearrangement, and the loss of a C 1 unit.Usually, the terpenoid unit of meroterpenoids follows the isoprene rule [e.g., centrapalus coumarin O (5)]; however, in compounds 2-4, it is not possible to sequence the isoprene subunits according to the rule because of a rearrangement.
The tested compounds, centrapalus coumarins N (2) and O (5), proved to have considerable and selective antiproliferative activity against ovarian cancer cells.Moreover,

Table 4 .
Antiproliferative effects of centrapalus coumarins N (2) and O (5) on human gynecological cancer cell lines and NIH/3T3 mouse fibroblasts.